concentrations of SHH C25II (0–1,000 ng/ml, R&D Systems) were applied to ventralize cells. After two weeks of differentiation, cells were passed as previously in the same CDM with 1,000 ng/ml SHH, and FGF4 (10 ng/ml) was used to help the specification of the serotonergic fate. From the fourth week, serotonergic progenitors were seeded onto PO- and laminin-coated glass coverslips and cultured in a neuronal differentiation medium (NDM) consisting of neurobasal with 1 × N2, 1 × B27, 1 × NEAA supplemented with 1 µg/ml laminin, 0.2 mM vitamin C (Tocris Bioscience), 2.5 µM DAPT (Tocris Bioscience), 10 ng/ml glial cell line–derived neurotrophic factor (GDNF), 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml insulin-like growth factor-I (IGF-I) and 1 ng/ml transforming growth factor β3 (all from PeproTech).
